Decades of research on the myriad of biological activities that the polyamines, putrescine, spermidine and spermine play in cellular processes have shown the profound role they play in life (Cohen, S. S., “A Guide to the Polyamines” 1998, Oxford University Press, New York). As polycations at physiological pH, they bind tightly to and strongly modulate the biological activities of all of the anionic cellular components. Specific and strong interactions have been associated with DNA and RNA together with their associated chromatin proteins (Tabor, H. et al. 1,4-Diaminobutane (putrescine), spermidine, and spermine. Ann Rev. Biochem. 1976, 45, 285-306; Matthews, H. R. Polyamines, chromatin structure and transcription. BioEssays, 1993, 15, 561-566). Specific interactions of multicationic polyamines with microtubules has been recently shown (Wolff, J. Promotion of Microtubule Assembly by Oligocations: Cooperativity between Charged Groups. Biochemistry, 1998, 37, 10722-10729; Webb, H. K. et al., 1-(N-Allylamino)-11-(N-ethylamino)-4,8-diazaundecanes alter tubulin polymerization. J Med. Chem. 1999 42(8):1415-21). Allosteric regulation of membrane-bound enzymes including acetylcholinesterase has been shown (Kossorotow, A. et al. Regulatory effects of polyamines on membrane-bound acetylcholinesterase. Biochem. J. 1974, 144, 21-27).
There have also been reports on the involvement of polyamines in the induction of apoptosis. Stefanelli and coworkers report that using HL60 human leukemia cells (Stefanelli, C. et al. Spermine causes caspase activation in leukaemia cells. FEBS Letters, 1998, 437, 233-236) or a cell-free model (Stefanelli, C. et al. Spermine triggers the activation of caspase-3 in a cell-free model of apoptosis. FEBS Letters, 1999, 451, 95-98), addition of spermine led to the induction of apoptosis. This process was characterized by the release of cytochrome c from mitochondria, the dATP-dependent processing of pro-caspase-3 and the onset of caspase activity. This caspase activation was not blocked by antioxidants or inhibition of polyamine oxidase by MDL 72527. Thus these workers hypothesize a physiological role for the polyamines in the transduction of a death message.
Due to its four positive charges at physiological pH, spermine is predominantly bound to cellular components and its free concentration in the cell is very low despite the high cellular content of this polyamine (Marton, L. J. et al. Polyamines as targets for therapeutic intervention. Annu. Rev. Pharmacol. Toxicol. 1995, 35, 55-91). Thus, spermine may have the characteristics of a damage-sensing molecule, since its free concentration may increase rapidly following insults to nucleic acids, membranes or other storage sites. This increase would be proportional to the extent of the damage and could transduce a death signal to the mitochondria.
Other workers have explored the toxic mechanisms of polyamines and polyamine analogs. Poulin and coworkers showed that the deregulation of polyamine transport in L1210 cells over-expressing ornithine decarboxylase (ODC) led to a lethal accumulation of spermidine (Poulin, R. et al. Induction of apoptosis by excessive polyamine accumulation in ornithine decarboxylase-overproducing L1210 cells. Biochem. J 1995, 311, 723-727). They showed that this lethal insult was due to the induction of apoptosis. Polyamine oxidation was not responsible for the apoptosis observed. Wallace and coworkers showed a similar non-oxidative lethal action of spermine in BHK-21/C13 cells (Brunton, V. G. et al. Mechanisms of spermine toxicity in baby-hamster kidney (BHK) cells. Biochem. J. 1991, 280, 193-198). They also showed that MDL 72527 exacerbated the toxic effects of spermine.
Packham and Cleveland showed that the forced expression of ODC in 32D.3 murine myeloid cells caused an apoptotic cell death following IL-3 withdrawal (Packhani, G. et al. Ornithine decarboxylase is a mediator of c-myc-induced apoptosis. Mol. Cell. Biol. 1994, 14, 5741-5747). ODC induced cell death in a dose-dependent fashion, and α-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC effectively blocked ODC-induced cell death. Gerner and coworkers, in a series of experiments with ODC over-expressing or polyamine transport regulation deficient cell lines, demonstrated that loss of feedback regulation on the polyamine transport system is sufficient to induce apoptosis (Xie, X. et al. Loss of intracellular putrescine pool-size regulation induces apoptosis. Exp. Cell Res. 1997, 230, 386-392). Loss of regulation of the tight feedback controls on putrescine levels caused the cells to undergo apoptosis in a putrescine dose-dependant manner.
Yanagawa and coworkers showed that the antiproliferative effects of hepatocyte growth factor (HGF) involved the induction of apoptosis via an increase in ODC activity with a resultant increase in intracellular polyamine levels (Yanagawa, K. et al. The antiproliferative effect of HGF on hepatoma cells involves induction of apoptosis with increase in intracellular polyamine concentration levels. Oncol. Rep. 1998, 5, 185-190). Addition of the ODC inhibitor DFMO reduced the levels of polyamines and inhibited the apoptotic effects of HGF. This inhibition of apoptotic effects was again reversed by the addition of exogenous polyamines to the cells. The above reports indicate a clear apoptotic effect upon loss of regulation of polyamine pool concentrations. It is also clear that these effects occurred through a non-oxidative mechanism.
A series of modified spermine analogs, typified by N1, N11-diethylnorspermine (BE-3,3,3 also known as DENSPM), have been shown to super-induce the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT) and to work partially through an oxidative mechanism (Casero, R. A. et al. Spermidine/spermine N1-acetyltransferase—the turning point in polyamine metabolism. FASEB J. 1993, 7, 653-661). Porter and coworkers explored the cellular responses to a series of these analogs and compared their cytotoxicity, induction of SSAT and effects on the cell cycle (Kramer, D. L. et al. Effects of novel spermine analogues on cell cycle progression and apoptosis in MALME-3M human melanoma cells. Cancer Res. 1997, 57, 5521-5527). They concluded that cytotoxicity could not be correlated with the level of SSAT induction by these analogs, which left open the possibility that additional mechanism(s) could be involved. With only small changes in the analog's structure, great variability was seen in the effects on the cell cycle.
Using related analogs, Hu and Pegg showed that the deregulated uptake of polyamine analogs by the polyamine transporter caused rapid induction of apoptosis (Hu, R-H. et al. Rapid induction of apoptosis by deregulated uptake of polyamine analogues. Biochem. J. 1997, 328, 307-316).
Certain dibenzylputrescine analogs have been shown to have anti-proliferative effects against human and rodent tumor cell lines. Frydman et al. describe the cytotoxicity against three squamous cell carcinoma lines (SCC-38, SCC-4Y and SCC-13Y) and a rat hepatoma cell line (H-4-II-E) of the N1, N4-dibenzyl analogs of 1,3-diaminopropane, putrescine and cadaverine (Aizencang, G. et al. Antiproliferative effects of N1, N4-dibenzylputrescine in human and rodent tumor cells. Cellular and Molecular Biology, 1998, 44 (4), 615-625 and U.S. Pat. No. 5,677,350). IC50 values of between 100 to 300 μM were found against these cell lines with the putrescine and cadaverine N1, N4-dibenzyl analogs. These researchers describe the classic hallmarks of cells undergoing apoptotic cell death: vacuole formation, decrease in size, changes in staining by trypan blue and adherence.
Frydman et al. also demonstrated that co-incubation with a specific polyamine oxidase inhibitor, N1, N4-bis(buta-2,3-dienyl)butanediamine (MDL 72527) caused a five-fold increase in the activity of the analogs. Although a moderate inhibition of [1,4-14C]-putrescine uptake was found (Kiapp=6.5+/−1.7 μM with N1, N4-dibenzylputrescine compared to Kmapp=5.2+/−0.6 μM for putrescine), even a ten-fold excess of putrescine over N1, N4-dibenzylputrescine could not abolish its cell growth inhibitory effect. Moderate reductions in levels of intracellular polyamines were measured after 72 h of drug treatment. These decreases in the polyamine levels are of minor significance in comparison to the decreases achieved with therapeutic approaches designed to deplete polyamines (see U.S. Pat. No. 6,172,261 B1).
Results from an in vivo antiproliferative study using N1, N4-dibenzylputrescine (U.S. Pat. No. 5,677,350), suggested great promise for these analogs. These studies showed significant reduction in the weights of the treated compared to control tumors. Nude mice aged four weeks were subcutaneously inoculated with rat hepatoma H-4-II-E cells (10×106 cells) or human melanoma II-B-Mel-J (5×106 cells) and allowed to develop for 15 to 24 days. Administration of 0.15% N1, N4-dibenzylputrescine in the drinking water over 10 weeks showed no toxic effects on the animals. Several key observations were made in conjunction with these experiments. As stated above, the treatment with N1, N4-dibenzylputrescine showed no liver or kidney damage following the treatment. Metastatic lung tumors that were observed in the control animals did not appear in the treated animals. Most importantly, the growth of the tumors was strongly inhibited by a factor of 6 or 7-fold in the treated animals. Further study showed no significant changes in the polyamine levels in the tumors from the treated in comparison to the control animals.
A recent report suggests an explanation for the increased cytotoxicity observed in the presence of MDL 72527 (Dai, H. et al. The polyamine oxidase inhibitor MDL-72,527 selectively induces apoptosis of transformed hematopoietic cells through lysosomotropic effects. Cancer Research, 1999, 59, 4944-4954). This compound, previously reported to be a relatively non-toxic, selective polyamine oxidase (PAO) inhibitor, was shown to induce apoptosis in transformed hematopoietic cells. It is interesting to note that this compound was non-toxic to primary myeloid progenitors. Cellular characterization of this compound revealed features strikingly similar to those reported for the dibenzylputrescine analogs above. Although this compound decreased the levels of putrescine and spermidine (it also increased the level of N1-acetylspermidine), these effects were expected based on the compound's action as an inhibitor of PAO. The cytotoxic effects of this compound were not blocked by co-treatment with exogenous putrescine or spermidine. These effects were also not influenced by over-expression or inhibition of ornithine decarboxylase (ODC), the rate-limiting polyamine biosynthetic enzyme. A well-characterized specific inhibitor of ODC, DFMO caused the increased uptake of MDL 72527 leading to greater cytotoxicity but treatment with putrescine/DFMO/MDL 72527 gave the same effects as MDL 72527 alone.
In summary, these reports showed that N1, N4-dibenzylputrescine and other similar analogs were not cytotoxic by depleting the intracellular polyamine levels. The fact that a specific and potent PAO inhibitor increased their activity suggested that a polyamine oxidase-mediated mechanism was not responsible. Despite this limited knowledge about the mechanism, these compounds did show moderate IC50 values against several different cancer cell lines. They also showed the hallmarks of compounds that operate through an apoptotic mechanism. N1, N4-dibenzylputrescine showed significant promise in a mouse xenograft anti-tumor model. This compound was orally active and showed no toxic effects even after a 40-day treatment. Additional advantages of these compounds have been their easy and inexpensive synthesis.
Mitochondria apparently play a major role in apoptotic pathways. It is now generally accepted that a decrease in the mitochondrial membrane potential is an early universal event of apoptosis (Mignotte, B. et al. Mitochondria and apoptosis. Eur. J Biochem. 1998, 252, 1-15). Mitochondria participate in the early steps of apoptosis, in response to many stimuli, through the release of cytochrome c into the cytoplasm. Recent literature reports indicate that many molecules, including several clinically promising agents, induce apoptosis through the release of cytochrome c from the mitochondria. One well-established mechanism for this release is the swelling of the mitochondrial inner membrane followed by rupture of the outer membrane/matrix. The release of the positively charged cytochrome c protein from the mitochondria is strongly linked to the induction of apoptosis (Green, D. R. et al. Mitochondria and Apoptosis. Science, 1998, 281, 1309-1312). The released cytochrome c initiates a complex pathway that ultimately results in the activation of caspase-3.
Tamanoi and coworkers showed that a set of four structurally diverse farnesyltransferase inhibitors induce the release of cytochrome c from mitochondria of ν-K-ras-transformed normal rat kidney cells (Suzuki, N. et al. Farnesyltransferase inhibitors induce cytochrome c release and caspase 3 activation preferentially in transformed cells. Proc. Natl. Acad. Sci. USA, 1998, 95, 15356-115361). They showed that this release resulted in caspase-3 activation and was observed preferentially in transformed cells compared to the normal cells.
Debatin and coworkers showed that betulinic acid, a melanoma-specific cytotoxic agent, triggered CD95 (APO-1/Fas)- and p53-independent apoptosis via release of cytochrome c and apoptosis inducing factor (AIF) from the mitochondria into the cytosol (Fulda, S. et al. Activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid. J. Biol. Chem. 1998, 273, 33942-33948). The fact that this drug-induced apoptosis (via a direct effect on mitochondria) did not involve two common resistance mechanisms suggests that betulinic acid may bypass some forms of drug resistance (Fulda, S. et al. Betulinic acid triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal tumors. Cancer Res. 1997, 57, 4956-4964).
An additional agent, presently in Phase III trials of metastatic breast and ovarian cancer, lonidamine (1-[(2,4-dichlorophenyl)methyl]-1H-indazole-3-carboxylic acid), also acts independently of p53 status via a direct action on the mitochondrial permeability transition pore (Ravagnan, L. et al. Lonidamine triggers apoptosis via a direct, Bcl-2-inhibited effect on the mitochondrial permeability transition pore. Oncogene 1999, 18, 2537-2546).
The early universal apoptotic event of a decrease in the mitochondrial membrane potential may occur by the opening of pores in the inner membrane of mitochondria. These pores allow the passage of compounds of molecular weight of <1500 Da through the membrane and several of these have been directly linked to the induction of apoptosis.
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